Compositions and methods for boosting the efficacy of adoptive cellular immunotherapy

ABSTRACT

The present disclosure provides compositions and methods for boosting, augmenting or enhancing the efficacy of the adoptive cellular immunotherapy by using modified T cells expressing an antigen binding protein in conjunction with modified cells (such as hematopoietic progenitor cells, modified human immune system cells or a combination thereof) expressing the antigen specifically bound by the antigen binding protein of the modified T cells.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application 62/069,168 filed Oct. 27, 2014, whichapplication is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant Nos.CA114536, AI053193, and P51-OD010425 awarded by the National Institutesof Health. The government has certain rights in this invention.

BACKGROUND

Technical Field

The present disclosure relates generally to compositions and methods forenhancing or boosting the efficacy of an immunotherapy. Morespecifically, the present disclosure relates to using a T cellexpressing an antigen binding protein (e.g., chimeric antigen receptor)in conjunction with cells (such as hematopoietic progenitor cells,modified human immune system cells or a combination thereof) modified toexpress the antigen specifically bound by the antigen binding protein ofthe modified T cells to boost adoptive cellular immunotherapy fortreating diseases or disorders associated with expression of thetargeted antigen, such as cancer.

Description of the Related Art

The introduction of tumor-targeting receptors into T cells by genetransfer allows the rapid generation of tumor-specific T cells from anycancer patient for adoptive T-cell therapy. A promising strategyinvolves engineering T cells with synthetic chimeric antigen receptors(CARs) comprised of a single chain antibody or other binding domain thatis specific for a tumor cell-surface molecule and is linked to one ormore T-cell signaling molecules (Turtle et al., Curr. Opin. Immunol.24:633, 2012; Barrett et al., Annu. Rev. Med. 65:10.1, 2014; Sadelain etal., Cancer Discovery 3:388, 2013). Recent trials using CAR-modified Tcells (CAR-T cells) specific for the CD19 molecule on B-cellmalignancies demonstrated marked tumor regression in a subset ofpatients with advanced disease (Barrett et al., 2014; Sadelain et al.,2013; Kalos et al., Sci. Transl. Med. 3:95ra73, 2011; Kochenderfer andRosenberg, Nat. Rev. Clin. Oncol. 10:267, 2013). Extending this therapyto common epithelial cancers poses several challenges, including theidentification of molecules expressed on tumor cells that can betargeted safely with T cells. This is underscored by serious and evenfatal toxicities that have been observed due to on-target/off-tumoreffects of CAR-therapy on normal cells that express the target molecule(Lamers et al., Mol. Ther. 21:904, 2013; Morgan et al., Mol. Ther.18:843, 2010). In addition, being able to identify the properties of Tcells that dictate their ability to survive and function in vivo remainimportant areas of research.

Clearly there is a need for alternative compositions and methods toenhance or boost adoptive cellular immunotherapies directed againstvarious cancers, such as leukemia and tumors. The presently disclosedembodiments address this need and provide other related advantages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show function and characterization of Rhesus macaque ROR1.(A) function of rhesus macaque T cells modified to express a R12-ROR1CAR with human or macaque 4-1BB and CD3ζ domains. (B) flow cytometricanalysis of purity and phenotype of ROR1 CAR-modified CD4⁺ and CD8⁺ Tcells using the tCD19 marker. The data shows the phenotype ofuntransduced T cells (mock), transduced T cells pre and post selectionon tCD19 and post selection on CD4 (top panels) or CD8 (bottom panels).All samples are gated on CD3⁺ cells. (C) cytolytic activity of CD4⁺ andCD8⁺ ROR1 CAR-T cells against ⁵¹Cr-labeled K562/ROR1 or K562 cells in a4-hour cytotoxicity assay. (D) Luminex cytokine assay of supernatantsobtained after 24 hours from triplicate co-cultures of 5×10⁴ ROR1 CAR-Tcells with K562/ROR1 cells or PMA/Ionomycin, or media alone as describedin Methods. Cytokine levels in the media control samples were below thedetection level. B-D, shows data from macaque A13002 that arerepresentative of independent experiments in 3 animals.

FIGS. 2A-2E show monitoring of toxicity and in vivo persistence of ROR1CAR-T cells. (A) body weight and serum chemistry before and at theindicated days after the T-cell infusion. The grey shaded area demarksthe rhesus macaque specific normal range for each parameter. (B) plasmacytokine levels were measured prior to and post-infusion using arhesus-specific multiplex cytokine assay. (C) PBMC were obtained beforeand at the indicated days after the infusion of ROR1 CAR- and controlEGFRt⁺ T cells. The frequency of transferred T cells (%) within theCD3⁺CD4⁺ subset and CD3⁺CD8⁺ subset was determined by flow cytometryafter staining with mAbs specific for CD3, CD4, CD8, and CD19 or forEGFRt. (D) absolute numbers of R12-ROR1 CAR⁺ and EGFRt⁺ T cells in theperipheral blood measured by flow cytometry and calculated based on theresults of a CBC on the indicated days in an accredited clinicallaboratory. (E) DNA was isolated from samples of PBMC obtained on theindicated days and examined by real-time qPCR (TaqMan) for the presenceof transgene vector-specific sequences.

FIGS. 3A-3D show in vivo migration and function of R12-ROR1 CAR-T cells.(A) BM and LN samples obtained prior to and on day 5 after the T-cellinfusion were stained with mAbs specific for CD3, CD4, CD8, and CD19 orfor EGFRt, and examined by flow cytometry after gating on CD3³⁰CD4⁺ orCD3 ⁺CD8⁺T cells. (B) detection of ROR1⁺ B-cell precursors in the BM.Samples of BM were obtained before and on day 5 post-infusion andexamined by flow cytometry for the presence of a ROR1-expressingCD19⁺CD45^(intermediate) B cell subset. (C) absolute number of CD19⁺ Bcells in the peripheral blood samples obtained before and after theT-cell infusion determined by staining with mAbs specific for CD19, CD3,CD4, and CD8 and flow cytometry to detect CD19⁺CD3⁻ B cells. Absolutenumbers were determined based on the lymphocyte count on a CBC obtainedat the same time and determined in an accredited clinical laboratory.(D) CD107A degranulation assay. PBMC were obtained before and at day 7post-infusion, stimulated ex vivo with K562/ROR1 cells, media, or PMAand Ionomycin, and examined by flow cytometry for expression of CD107Aas described in Methods. Cultured ROR1 CAR-T cells served as positivecontrol.

FIGS. 4A-4E show persistence, migration, and safety of ROR1 CAR-T cellsgiven at a high cell dose. (A) schematic overview of the T cellinfusions. Autologous ROR1 CAR-T cells were adoptively transferred at atotal dose of 5×10⁸/kg. Control tCD34⁺ gene-marked T cells wereadministered at an equivalent dose. Samples of BM and LN were obtainedbefore and on day 3 after the T-cell infusion. (B) flow cytometricanalysis of PBMC obtained from macaques A13011 and A13002 before and onday 1 the T-cell infusion. (C) The frequency of transferred T cells (%)within the CD3⁺CD4⁺ subset and CD3⁺CD8⁺ subset was determined afterstaining with mAbs specific for CD3, CD4, CD8, and CD19 or for tCD34. C,PBMC, BM, and LN samples were obtained from both macaques on day 3 afterthe T-cell infusion, stained with mAbs specific for CD3, CD4, CD8, andCD19 or tCD34, and examined by flow cytometry after gating on CD3⁺CD4⁺or CD3⁺CD8⁺ cells. The frequency of ROR1 CAR and control tCD34 marked Tcells in each subset is shown in the bar graphs for each animal. (D)Frequency of a ROR1 B-cell precursors in the BM before and after ROR1CAR-T cells. Samples of BM obtained before and 3 days after transfer ofROR1 CAR-T cells were examined by flow cytometry for the presence ofROR1-expressing CD19⁺CD45^(intermediate) B-cells. Shown arerepresentative stainings of macaque A13011 gated on CD19⁺ cells. (E)Frequency of ROR1⁺ B cells in the LN. Samples of LN were obtained beforeand 3 days after the T-cell infusion, stained with mAbs specific toCD19, CD45, and ROR1 or isotype and examined by flow cytometry for thepresence of ROR1⁺ CD45⁺ cells within the CD19⁺ subset. Shown are datafrom macaque A13011.

FIGS. 5A-5D show the effect of ROR1+ T cell (T-APC) challenge ontransferred ROR1 CAR-T cells in vivo. (A) Samples of PBMC were obtainedfrom A13011 and A13002 at the indicated days and examined by flowcytometry after staining with mAbs specific for CD3, CD4, CD8, and CD19or for CD34. The CBC was determined in an accredited clinicallaboratory. Shown are the absolute numbers of ROR1 CAR⁺ and tCD34⁺ Tcells in the peripheral blood in the CD3⁺CD4⁺ and CD3⁺CD8⁺ subset. (B)fold change in the absolute numbers of ROR1 CAR-T cells in the bloodafter T-APC challenge. The number of CD3⁺CD8⁺ T cells and CD19⁺CD8⁺ Tcells/μL of blood was measured by flow cytometry before and on indicateddays after the T-APC administration. The data show the fold-increaseover baseline of the absolute numbers of endogenous non-marked CD4⁺ andCD8⁺ T cells, R12 ROR1 CAR CD4⁺ and CD8⁺ T cells, and tCD34-marked CD4⁺and CD8⁺ T cells after T-APC administration. (C) presence of tROR1⁺ Tcells before and after the T-APC infusion in PBMC samples obtained atdays 1, 5, and 7 from an animal (A13011) with persisting ROR1 CAR-Tcells. PBMC were stained with anti-CD3 and anti-ROR1 mAbs. (D) presenceof tROR1⁺ T cells before and after the T-APC infusion in PBMC samplesobtained at days 1, 5, and 7 from an animal (A12022) without ROR1 CAR-Tcells.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides compositions and methodsfor boosting, augmenting or enhancing the efficacy of adoptive cellularimmunotherapy or treating a hyperproliferative disorder by administeringto human subject an effective amount of a composition comprising apopulation of modified human T cells comprising a nucleic acid moleculethat encodes an antigen binding protein (e.g., chimeric antigenreceptor, CAR), wherein the antigen binding protein comprises ahydrophobic portion disposed between an extracellular binding componentand an intracellular effector component; and a population of modifiedhuman hematopoietic progenitor cells, modified human immune system cellsor a combination thereof comprising a nucleic acid molecule that encodesthe antigen specifically recognized by the extracellular bindingcomponent of the antigen binding protein.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means ±20% of theindicated range, value, or structure, unless otherwise indicated. Itshould be understood that the terms “a” and “an” as used herein refer to“one or more” of the enumerated components. The use of the alternative(e.g., “or”) should be understood to mean either one, both, or anycombination thereof of the alternatives. As used herein, the terms“include,” “have” and “comprise” are used synonymously, which terms andvariants thereof are intended to be construed as non-limiting.

In addition, it should be understood that the individual compounds, orgroups of compounds, derived from the various combinations of thestructures and substituents described herein, are disclosed by thepresent application to the same extent as if each compound or group ofcompounds was set forth individually. Thus, selection of particularstructures or particular substituents is within the scope of the presentdisclosure.

The term “consisting essentially of” limits the scope of a claim to thespecified materials or steps, or to those that do not materially affectthe basic characteristics of a claimed invention. For example, a proteindomain, region, or module (e.g., a binding domain, hinge region, linkermodule) or a protein (which may have one or more domains, regions, ormodules) “consists essentially of” a particular amino acid sequence whenthe amino acid sequence of a domain, region, module, or protein includesextensions, deletions, mutations, or a combination thereof (e.g., aminoacids at the amino- or carboxy-terminus or between domains) that, incombination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%,5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, orprotein and do not substantially affect (i.e., do not reduce theactivity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%,10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), orprotein (e.g., the target binding affinity of a binding protein).

As used herein, a “hematopoietic progenitor cell” is a cell that can bederived from hematopoietic stem cells or fetal tissue and is capable offurther differentiation into mature cells types (e.g., immune systemcells). Exemplary hematopoietic progenitor cells include those with aCD24^(Lo) Lin⁻ CD117⁺ phenotype or those found in the thymus (referredto as progenitor thymocytes).

“Hematopoietic stem cells” refer to undifferentiated hematopoietic cellsthat are capable of self-renewal either in vivo, essentially unlimitedpropagation in vitro, and capable of differentiation to other cell typesincluding cells of the T cell lineage. Hematopoietic stem cells may beisolated, for example, from fetal liver, bone marrow, cord blood.

“Embryonic stem cells” or “ES cells” or “ESCs” refer to undifferentiatedembryonic stem cells that have the ability to integrate into and becomepart of the germ line of a developing embryo. Embryonic stem cells arecapable of differentiating into hematopoietic progenitor cells, and anytissue or organ. Embryonic stem cells that are suitable for use hereininclude cells from the J1 ES cell line, 129J ES cell line, murine stemcell line D3 (American Type Culture Collection), the R1 or E14K celllines derived from 129/Sv mice, cell lines derived from Balb/c andC57B1/6 mice, and human embryonic stem cells (e.g., from WiCell ResearchInstitute, WI; or ES cell International, Melbourne, Australia).

As used herein, an “immune system cell” means any cell of the immunesystem that originate from a hematopoietic stem cell in the bone marrow,which gives rise to two major lineages, a myeloid progenitor cell (whichgive rise to myeloid cells such as monocytes, macrophages, dendriticcells, meagakaryocytes and granulocytes) and a lymphoid progenitor cell(which give rise to lymphoid cells such as T cells, B cells and naturalkiller (NK) cells). Exemplary immune system cells include CD4+ T cells,CD8+ T cells, CD4− CD8− double negative T cells, γδ T cells, regulatoryT cells, natural killer cells, and dendritic cells. Macrophages anddendritic cells may be referred to as “antigen presenting cells” or“APCs,” which are specialized cells that can activate T cells when amajor histocompatibility complex (MHC) receptor on the surface of theAPC interacts with a TCR on the surface of a T cell. Alternatively, anyhematopoietic stem cell or immune system cell can be converted into anAPC by introducing a nucleic acid molecule that expresses an antigenrecognized by the TCR or by another antigen binding protein (e.g., CAR)

As used herein, the term “host” refers to a cell (e.g., T cell,hematopoietic progenitor cell) or microorganism targeted for geneticmodification with a heterologous or exogenous nucleic acid molecule toproduce a polypeptide of interest (e.g., cancer antigen-specific CAR).In certain embodiments, a host cell may optionally already possess or bemodified to include other genetic modifications that confer desiredproperties related or unrelated to biosynthesis of the heterologous orexogenous protein (e.g., inclusion of a detectable marker; deleted,altered or truncated CD34; increased co-stimulatory factor expression).In certain embodiments, a host cell is a human hematopoietic progenitorcell transduced with heterologous or exogenous nucleic acid moleculeencoding an antigen associated with disease and specifically bound by anantigen binding protein (e.g., CAR). A “T cell” is an immune system cellthat matures in the thymus and produces T cell receptors (TCRs). T cellscan be naïve (not exposed to antigen; increased expression of CD62L,CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45ROas compared to T_(CM)), memory T cells (T_(M)) (antigen-experienced andlong-lived), and effector cells (antigen-experienced, cytotoxic). T_(M)can be further divided into subsets of central memory T cells (T_(CM),increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, anddecreased expression of CD54RA as compared to naïve T cells) andeffector memory T cells (T_(EM), decreased expression of CD62L, CCR7,CD28, CD45RA, and increased expression of CD127 as compared to naïve Tcells or T_(CM)). Effector T cells (T_(E)) refers to aantigen-experienced CD8+ cytotoxic T lymphocytes that has decreasedexpression of CD62L ,CCR7, CD28, and are positive for granzyme andperforin as compared to T_(CM).

A “binding component” (also referred to as a “binding region” or“binding moiety”), as used herein, refers to a peptide, oligopeptide,polypeptide, or protein that possesses the ability to specifically andnon-covalently associate, unite, or combine with a target molecule(e.g., CD19, CD20, EGFRvIII, GD2, MUC16, ROR1, mesothelin, PD-L1, PD-L2,PSMA, cancer-associated neoantigen). A binding domain includes anynaturally occurring, synthetic, semi-synthetic, or recombinantlyproduced binding partner for a biological molecule, a molecular complex(i.e., complex comprising two or more biological molecules), or othertarget of interest. Exemplary binding domains include single chainimmunoglobulin variable regions (e.g., scTCR, scFv), receptorectodomains, ligands (e.g., cytokines, chemokines), or syntheticpolypeptides selected for the specific ability to bind to a biologicalmolecule, a molecular complex or other target of interest.

As used herein, “specifically binds” or “specific for” refers to anassociation or union of a binding protein (e.g., CAR or TCR) or abinding component (or fusion protein thereof) to a target molecule withan affinity or K_(a) (i.e., an equilibrium association constant of aparticular binding interaction with units of 1/M) equal to or greaterthan 10⁵ M⁻¹ (which equals the ratio of the on-rate [k_(on)] to theoff-rate [k_(off)] for this association reaction), while notsignificantly associating or uniting with any other molecules orcomponents in a sample. Binding proteins or binding domains (or fusionproteins thereof) may be classified as “high affinity” binding proteinsor binding domains (or fusion proteins thereof) or as “low affinity”binding proteins or binding domains (or fusion proteins thereof). “Highaffinity” binding proteins or binding domains refer to those bindingproteins or binding domains having a K_(a) of at least 10⁷ M⁻¹, at least10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, atleast 10 ¹² M⁻¹, or at least 10¹³ M⁻¹. “Low affinity” binding proteinsor binding domains refer to those binding proteins or binding domainshaving a K_(a) of up to 10⁷ M⁻¹, up to 10⁶ M⁻¹, up to 10⁵ M⁻¹.Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(d)) of a particular binding interaction with units of M(e.g., 10⁻⁵ M to 10⁻¹³ M).

A variety of assays are known for identifying binding domains of thepresent disclosure that specifically bind a particular target, as wellas determining binding domain or fusion protein affinities, such asWestern blot, ELISA, analytical ultracentrifugation, spectroscopy andsurface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002;Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173,5,468,614, or the equivalent).

As used herein, a “hinge region” or a “hinge” refers to (a) animmunoglobulin hinge sequence (made up of, for example, upper and coreregions) or a functional variant thereof, (b) a type II C-lectininterdomain (stalk) region or a functional variant thereof, or (c) acluster of differentiation (CD) molecule stalk region or a functionalvariant thereof. As used herein, a “wild type immunoglobulin hingeregion” refers to a naturally occurring upper and middle hinge aminoacid sequences interposed between and connecting the CH1 and CH2 domains(for IgG, IgA, and IgD) or interposed between and connecting the CH1 andCH3 domains (for IgE and IgM) found in the heavy chain of an antibody.In certain embodiments, a hinge region is human, and in particularembodiments, comprises a human IgG hinge region.

A “hydrophobic portion,” as used herein, means any amino acid sequencehaving a three-dimensional structure that is thermodynamically stable ina cell membrane, and generally ranges in length from about 15 aminoacids to about 30 amino acids. The structure of a hydrophobic domain maycomprise an alpha helix, a beta barrel, a beta sheet, a beta helix, orany combination thereof.

As used herein, an “effector component” or “effector domain” is anintracellular portion of a fusion protein or receptor that can directlyor indirectly promote a biological or physiological response in a cellwhen receiving the appropriate signal. In certain embodiments, aneffector component is part of a protein, fusion protein or proteincomplex that receives a signal when bound, or it binds directly to atarget molecule, which triggers a signal from the effector domain. Aneffector domain may directly promote a cellular response when itcontains one or more signaling domains or motifs, such as animmunoreceptor tyrosine-based activation motif (ITAM). In otherembodiments, an effector component will indirectly promote a cellularresponse by associating with one or more other proteins that directlypromote a cellular response.

A “linker” refers to an amino acid sequence that connects two proteins,polypeptides, peptides, domains, regions, or motifs and may provide aspacer function compatible with interaction of the two sub-bindingdomains so that the resulting polypeptide retains a specific bindingaffinity (e.g., scTCR) to a target molecule or retains signalingactivity (e.g., TCR complex). In certain embodiments, a linker iscomprised of about two to about 35 amino acids, for instance, or aboutfour to about 20 amino acids or about eight to about 15 amino acids orabout 15 to about 25 amino acids. In further embodiments, a linker is avariable region linker that connects a heavy chain immunoglobulinvariable region to a light chain immunoglobulin variable region orconnects T cell receptor V_(aαβ) and C_(α/β) chains (e.g., V_(α)-C_(α),V_(β)-C_(β), V_(α)-V_(β)) or connects each V_(α)-C_(α), V_(β)-C_(β),V_(α)-V_(β)pair to a hinge or hydrophobic domain.

“Junction amino acids” or “junction amino acid residues” refer to one ormore (e.g., about 2-10) amino acid residues between two adjacent motifs,regions or domains of a polypeptide, such as between a binding domainand an adjacent constant domain or between a TCR chain and an adjacentself-cleaving peptide. Junction amino acids may result from theconstruct design of a fusion protein (e.g., amino acid residuesresulting from the use of a restriction enzyme site during theconstruction of a nucleic acid molecule encoding a fusion protein).

An “altered component” or “altered domain” or “altered protein” refersto a motif, region, domain, peptide, polypeptide, or protein with asequence identity to a wild type motif, region, domain, peptide,polypeptide, or protein (e.g., wild type intracellular domains CD3ζ,CD134, CD137) of at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

As used herein, an “antigen” encoded by nucleic acid molecule containedin a modified human hematopoietic progenitor cells, modified humanimmune system cells or a combination thereof refers to any biologicalmolecule (e.g., protein, carbohydrate) that is associated with a diseaseor disorder (e.g., cancer) and targeted for immunotherapy. In certainembodiments, antigen may be an anti-idiotype antibody or anti-idiotypeantibody binding fragment thereof, or an antibody or antibody bindingfragment thereof specific for an antigen binding protein (e.g., CAR)expressed by a modified T cell. Exemplary antigens include α-fetoprotein(AFP), B7H4, BTLA, CD3, CD19, CD20, CD25, CD22, CD28, CD30, CD40,CD44v6, CD52, CD56, CD79b, CD80, CD81, CD86, CD134 (OX40), CD137(4-1BB), CD151, CD276, CA125, CEA, CEACAM6, c-Met, CT-7, CTLA-4, EGFR,EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, FLT1, FLT4, Frizzled,O-acetyl-GD2, GD2, GHRHR, GHR, GITR, gp130, HVEM, IGF1R, IL6R, KDR,L1CAM, Lewis A, Lewis Y, LTβR, LIFRβ, LRP5, MAGE, mesothelin, MUC1,NY-ESO-1, a cancer-specific neoantigen, OSMRβ, PD1, PD-L1, PD-L2, PSMA,PTCH1, RANK, Robol, ROR1, TERT, TGFBR2, TGFBR1, TLR7, TLR9, TNFRSF4,TNFR1, TNFR2, tyrosinase, TWEAK-R, or WT-1.

“T cell receptor” (TCR) refers to an immunoglobulin superfamily member(having a variable binding domain, a constant domain, a transmembraneregion, and a short cytoplasmic tail; see, e.g., Janeway et al.,Immunobiology: The Immune System in Health and Disease, 3^(rd) Ed.,Current Biology Publications, p. 4:33, 1997) capable of specificallybinding to an antigen peptide bound to a MHC receptor. A TCR can befound on the surface of a cell or in soluble form and generally iscomprised of a heterodimer having α and β chains (also known as TCRα andTCRα, respectively), or γ and δ chains (also known as TCRγ and TCRδ,respectively). Like immunoglobulins, the extracellular portion of TCRchains (e.g., α-chain, β-chain) contain two immunoglobulin domains, avariable domain (e.g., α-chain variable domain or V_(a), β-chainvariable domain or V_(β); typically amino acids 1 to 116 based on Kabatnumbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5^(th) ed.) at the N-terminus, andone constant domain (e.g., α-chain constant domain or C_(α) typicallyamino acids 117 to 259 based on Kabat, β-chain constant domain or C_(β),typically amino acids 117 to 295 based on Kabat) adjacent to the cellmembrane. Also like immunoglobulins, the variable domains containcomplementary determining regions (CDRs) separated by framework regions(FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138,1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al.,Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, a TCR is foundon the surface of T cells (or T lymphocytes) and associates with the CD3complex. The source of a TCR as used in the present disclosure may befrom various animal species, such as a human, mouse, rat, rabbit orother mammal.

“CD3” is known in the art as a multi-protein complex of six chains (see,Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). Inmammals, the complex comprises a CD3γ chain, a CD3δ chain, two CD3εchains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, and CD3ε chainsare highly related cell surface proteins of the immunoglobulinsuperfamily containing a single immunoglobulin domain. The transmembraneregions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, whichis a characteristic that allows these chains to associate with thepositively charged T cell receptor chains. The intracellular tails ofthe CD3γ, CD3δ, and CD3ε chains each contain a single conserved motifknown as an immunoreceptor tyrosine-based activation motif or ITAM,whereas each CD3δ chain has three. Without wishing to be bound bytheory, it is believed the ITAMs are important for the signalingcapacity of a TCR complex. CD3 as used in the present disclosure may befrom various animal species, including human, mouse, rat, or othermammals.

As used herein, “TCR complex” refers to a complex formed by theassociation of C3 with TCR. For example, a TCR complex can be composedof a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of CD3ζchains, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex canbe composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimerof CD3δ chains, a TCRγ chain, and a TCRδ chain.

A “component of a TCR complex,” as used herein, refers to a TCR chain(i.e., TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε orCD3ζ), or a complex formed by two or more TCR chains or CD3 chains(e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complexof CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex ofTCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

As used herein, “nucleic acid” or “nucleic acid molecule” refers to anyof deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated, for example, by the polymerasechain reaction (PCR) or by in vitro translation, and fragments generatedby any of ligation, scission, endonuclease action, or exonucleaseaction. In certain embodiments, the nucleic acids of the presentdisclosure are produced by PCR. Nucleic acids may be composed ofmonomers that are naturally occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), analogs of naturallyoccurring nucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havemodifications in or replacement of sugar moieties, or pyrimidine orpurine base moieties. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. Nucleic acid moleculescan be either single stranded or double stranded.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacid could be part of a vector and/or such nucleic acid or polypeptidecould be part of a composition (e.g., a cell lysate), and still beisolated in that such vector or composition is not part of the naturalenvironment for the nucleic acid or polypeptide. The term “gene” meansthe segment of DNA involved in producing a polypeptide chain; itincludes regions preceding and following the coding region “leader andtrailer” as well as intervening sequences (introns) between individualcoding segments (exons).

As used herein, the term “recombinant” refers to a cell, microorganism,nucleic acid molecule, or vector that has been modified by introductionof an exogenous nucleic acid molecule, or refers to a cell ormicroorganism that has been altered such that expression of anendogenous nucleic acid molecule or gene is controlled, deregulated orconstitutive, where such alterations or modifications may be introducedby genetic engineering. Genetic alterations may include, for example,modifications introducing nucleic acid molecules (which may include anexpression control element, such as a promoter) encoding one or moreproteins or enzymes, or other nucleic acid molecule additions,deletions, substitutions, or other functional disruption of or additionto a cell's genetic material. Exemplary modifications include those incoding regions or functional fragments thereof of heterologous orhomologous polypeptides from a reference or parent molecule.

As used herein, “mutation” refers to a change in the sequence of anucleic acid molecule or polypeptide molecule as compared to a referenceor wild-type nucleic acid molecule or polypeptide molecule,respectively. A mutation can result in several different types of changein sequence, including substitution, insertion or deletion ofnucleotide(s) or amino acid(s). In certain embodiments, a mutation is asubstitution of one or three codons or amino acids, a deletion of one toabout 5 codons or amino acids, or a combination thereof.

A “conservative substitution” is recognized in the art as a substitutionof one amino acid for another amino acid that has similar properties.Exemplary conservative substitutions are well known in the art (see,e.g., WO 97/09433 at page 10; Lehninger, Biochemistry, 2^(nd) Edition;Worth Publishers, Inc. NY, N.Y., pp.71-77, 1975; Lewin, Genes IV, OxfordUniversity Press, NY and Cell Press, Cambridge, Mass., p. 8, 1990).

The term “construct” refers to any polynucleotide that contains arecombinant nucleic acid. A construct may be present in a vector (e.g.,a bacterial vector, a viral vector) or may be integrated into a genome.A “vector” is a nucleic acid molecule that is capable of transportinganother nucleic acid. Vectors may be, for example, plasmids, cosmids,viruses, a RNA vector or a linear or circular DNA or RNA molecule thatmay include chromosomal, non-chromosomal, semi-synthetic or syntheticnucleic acids. Exemplary vectors are those capable of autonomousreplication (episomal vector) or expression of nucleic acids to whichthey are linked (expression vectors).

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabiesand vesicular stomatitis virus), paramyxovirus (e.g., measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include avian leukosis-sarcoma, mammalianC-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

“Lentiviral vector,” as used herein, means HIV-based lentiviral vectorsfor gene delivery, which can be integrative or non-integrative, haverelatively large packaging capacity, and can transduce a range ofdifferent cell types. Lentiviral vectors are usually generated followingtransient transfection of three (packaging, envelope and transfer) ormore plasmids into producer cells. Like HIV, lentiviral vectors enterthe target cell through the interaction of viral surface glycoproteinswith receptors on the cell surface. On entry, the viral RNA undergoesreverse transcription, which is mediated by the viral reversetranscriptase complex. The product of reverse transcription is adouble-stranded linear viral DNA, which is the substrate for viralintegration into the DNA of infected cells.

The term “operably-linked” refers to the association of two or morenucleic acid molecules on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably-linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., the coding sequence isunder the transcriptional control of the promoter). “Unlinked” meansthat the associated genetic elements are not closely associated with oneanother and the function of one does not affect the other.

As used herein, “expression vector” refers to a DNA construct containinga nucleic acid molecule that is operably-linked to a suitable controlsequence capable of effecting the expression of the nucleic acidmolecule in a suitable host. Such control sequences include a promoterto effect transcription, an optional operator sequence to control suchtranscription, a sequence encoding suitable mRNA ribosome binding sites,and sequences which control termination of transcription andtranslation. The vector may be a plasmid, a phage particle, a virus, orsimply a potential genomic insert. Once transformed into a suitablehost, the vector may replicate and function independently of the hostgenome, or may, in some instances, integrate into the genome itself. Inthe present specification, “plasmid,” “expression plasmid,” “virus” and“vector” are often used interchangeably.

The term “expression”, as used herein, refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid molecule may be incorporated into the genome of a cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein, “heterologous” or “exogenous” nucleic acid molecule,construct or sequence refers to a nucleic acid molecule or portion of anucleic acid molecule that is not native to a host cell, but may behomologous to a nucleic acid molecule or portion of a nucleic acidmolecule from the host cell. The source of the heterologous or exogenousnucleic acid molecule, construct or sequence may be from a differentgenus or species. In certain embodiments, a heterologous or exogenousnucleic acid molecule is added (i.e., not endogenous or native) to ahost cell or host genome by, for example, conjugation, transformation,transfection, electroporation, or the like, wherein the added moleculemay integrate into the host genome or exist as extra-chromosomal geneticmaterial (e.g., as a plasmid or other form of self-replicating vector),and may be present in multiple copies. In addition, “heterologous”refers to a non-native enzyme, protein or other activity encoded by anexogenous nucleic acid molecule introduced into the host cell, even ifthe host cell encodes a homologous protein or activity.

As described herein, more than one heterologous or exogenous nucleicacid molecule can be introduced into a host cell as separate nucleicacid molecules, as a plurality of individually controlled genes, as apolycistronic nucleic acid molecule, as a single nucleic acid moleculeencoding a fusion protein, or any combination thereof. For example, asdisclosed herein, a host cell can be modified to express two or moreheterologous or exogenous nucleic acid molecules encoding desiredantigen-specific binding proteins (e.g., CAR, TCRα and TCRβ). When twoor more exogenous nucleic acid molecules are introduced into a hostcell, it is understood that the two more exogenous nucleic acidmolecules can be introduced as a single nucleic acid molecule (e.g., ona single vector), on separate vectors, integrated into the hostchromosome at a single site or multiple sites. The number of referencedheterologous nucleic acid molecules or protein activities refers to thenumber of encoding nucleic acid molecules or the number of proteinactivities, not the number of separate nucleic acid molecules introducedinto a host cell.

As used herein, the term “endogenous” or “native” refers to a gene,protein, or activity that is normally present in a host cell. Moreover,a gene, protein or activity that is mutated, overexpressed, shuffled,duplicated or otherwise altered as compared to a parent gene, protein oractivity is still considered to be endogenous or native to thatparticular host cell. For example, an endogenous control sequence from afirst gene (e.g., promoter, translational attenuation sequences) may beused to alter or regulate expression of a second native gene or nucleicacid molecule, wherein the expression or regulation of the second nativegene or nucleic acid molecule differs from normal expression orregulation in a parent cell.

The term “homologous” or “homolog” refers to a molecule or activityfound in or derived from a host cell, species or strain. For example, aheterologous or exogenous nucleic acid molecule may be homologous to anative host cell gene, and may optionally have an altered expressionlevel, a different sequence, an altered activity, or any combinationthereof.

“Sequence identity,” as used herein, refers to the percentage of aminoacid residues in one sequence that are identical with the amino acidresidues in another reference polypeptide sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. The percentage sequenceidentity values can be generated using the NCBI BLAST2.0 software asdefined by Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402, with the parameters set to default values.

As used herein, “hyperproliferative disorder” refers to excessive growthor proliferation as compared to a normal or undiseased cell. Exemplaryhyperproliferative disorders include tumors, cancers, neoplastic tissue,carcinoma, pre-malignant cells, as well as non-neoplastic ornon-malignant hyperproliferative disorders (e.g., adenoma, fibroma,lipoma, leiomyoma, hemangioma, restenosis, as well as autoimmunediseases such as rheumatoid arthritis, osteoarthritis, psoriasis,inflammatory bowel disease, or the like).

Modified Cells Expressing Antigen Binding Protein or Antigen

In certain aspects, the instant disclosure provides an adoptive cellularimmunotherapy composition, comprising a population of modified humanhematopoietic progenitor cells, modified human immune system cells or acombination thereof, wherein a first population of modified cells are Tcells comprising a nucleic acid molecule that encodes an antigen bindingprotein (e.g., CAR), and a second population of modified cellscomprising a nucleic acid molecule that encodes the antigen (e.g.,cancer-specific antigen, anti-idiotypic antibody or binding fragmentthereof). In these embodiments, the antigen binding protein comprises ahydrophobic portion disposed between an extracellular binding componentand an intracellular effector component, and the extracellular bindingcomponent is specific for the antigen encoded by the second populationof modified cells. For example, an antigen binding protein may be a Tcell receptor (TCR) or a chimeric antigen receptor.

An advantage of these compositions is that a lower dose of, for example,an antigen-specific CAR T cell may be administered to a patient, andthen administering (e.g., simultaneously or sequentially) a secondpopulation of cells expressing the antigen recognized by the CAR willboost the effect of the adoptive immunotherapy. Another advantage isthat the boosting effect can also improve homing so the cells migrate tothe tissue of interest. Yet another advantage of the combination of aCAR expressing T cell and an artificial APC is to systemically boostingthe effect of the adoptive immunotherapy in order to get more activecells at the site of the tumor.

In certain other aspects, a modified hematopoietic progenitor cellcomprising a nucleic acid molecule that encodes the antigen isadministered to a subject, wherein the encoded antigen is under thecontrol of a regulated promoter. The modified hematopoietic progenitorcell will locate in a tissue of interest and replicate (e.g., bonemarrow or lymph nodes). Then, the expression of antigen in the expandedmodified hematopoietic progenitor cells may be induced simultaneous withor just after the subject is administered a first population of modifiedcells T cells comprising a nucleic acid molecule that encodes an antigenbinding protein (e.g., CAR), wherein the number of introduced “APCs” ismuch greater than would be available by ex vivo expansion andadministration. In certain embodiments, the administered secondpopulation of cells is irradiated before administration.

In any of the aforementioned embodiments, the antigen binding proteincomprises binding component, a hydrophobic portion and an intracellulareffector component. For example the binding component may be an antibodyvariable fragment (Fv), a TCR variable domain, a receptor ectodomain, ora ligand. In further embodiments, the binding component is a scFv orscTCR comprising a variable region linker, such as a linker comprises a(Gly_(x)Ser_(y))_(n), wherein x and y are independently an integer from1 to 5, and n is an integer from 1 to 10. In further embodiments, thebinding component is specific for α-fetoprotein (AFP), B7H4, BTLA, CD3,CD19, CD20, CD25, CD22, CD28, CD30, CD40, CD44v6, CD52, CD56, CD79b,CD80, CD81, CD86, CD134 (OX40), CD137 (4-1BB), CD151, CD276, CA125, CEA,CEACAM6, c-Met, CT-7, CTLA-4, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4,EphA2, FLT1, FLT4, Frizzled, O-acetyl-GD2, GD2, GHRHR, GHR, GITR, gp130,HVEM, IGF1R, IL6R, KDR, L1CAM, Lewis A, Lewis Y, LTβR, LIFRβ, LRP5,MAGE, mesothelin, MUC1, NY-ESO-1, a cancer-specific neoantigen, OSMRβ,PD1, PD-L1, PD-L2, PSMA, PTCH1, RANK, Robo1, ROR1, TERT, TGFBR2, TGFBR1,TLR7, TLR9, TNFRSF4, TNFR1, TNFR2, tyrosinase, TWEAK-R, or WT-1.

In still further embodiments, the hydrophobic portion is a transmembranedomain, such as a CD4, CD8, CD28 or CD27 transmembrane domain.

In certain embodiments, an intracellular effector component comprises anintracellular region of CD38, CD36, CD3C, CD25, CD27, CD28, CD79A,CD79B, CD134, CD137, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS,Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk,SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combinationthereof. In particular embodiments, the intracellular effector componentcomprises CD3ζ and one or more of CD27, CD28, CD134, and CD137, or theintracellular effector component comprises LRP, NOTCH1, NOTCH2, NOTCH3,NOTCH4, ROR2, or Ryk.

In any of the aforementioned embodiments, the modified cell is an immunesystem cell, such as a CD4+ T cell, a CD8+ T cell, a CD4− CD8− doublenegative T cell, a γδ T cell, regulatory T cell, a natural killer cell,a dendritic cell, or any combination thereof. In certain embodiments,the T cell is a naïve T cell, a central memory T cell, an effectormemory T cell, or any combination thereof For example, the firstpopulation of modified T cells may consist essentially of CD4+ T cells,CD8+ T cells, or both CD4+ and CD8+ T cells, and the second populationof modified cells comprises modified human hematopoietic progenitorcells. In other examples, the first population of modified T cells mayconsist essentially of CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+T cells, and the second population of modified cells comprises modifiedhuman immune system cells, such as cells consisting essentially of CD4+T cells, a CD8+ T cells, or both CD4+ and CD8+ T cells.

In any of the aforementioned embodiments, the modified cell populationsare recombinantly modified ex vivo by use of, for example, a viralvector, such as a lentiviral vector or a γ-retroviral vector. In furtherembodiments, the cell populations being modified are syngeneic,allogeneic, or autologous cells. In any of the aforementionedembodiments, the modified cell populations are further formulated with apharmaceutically acceptable carrier, diluent, or excipient as describedherein.

A variety of criteria known to persons skilled in the art indicatewhether an amino acid that is substituted at a particular position in apeptide or polypeptide is conservative (or similar). For example, asimilar amino acid or a conservative amino acid substitution is one inwhich an amino acid residue is replaced with an amino acid residuehaving a similar side chain. Similar amino acids may be included in thefollowing categories: amino acids with basic side chains (e.g., lysine,arginine, histidine); amino acids with acidic side chains (e.g.,aspartic acid, glutamic acid); amino acids with uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, histidine); amino acids with nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); amino acids with beta-branched side chains(e.g., threonine, valine, isoleucine), and amino acids with aromaticside chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, whichis considered more difficult to classify, shares properties with aminoacids that have aliphatic side chains (e.g., leucine, valine,isoleucine, and alanine). In certain circumstances, substitution ofglutamine for glutamic acid or asparagine for aspartic acid may beconsidered a similar substitution in that glutamine and asparagine areamide derivatives of glutamic acid and aspartic acid, respectively. Asunderstood in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide (e.g., using GENEWORKS, Align, the BLAST algorithm, or otheralgorithms described herein and practiced in the art).

In certain embodiments, an antigen specific binding protein a bindingcomponent that is at least about 90% or at least about 95% identical toan amino acid sequence to the original wild type sequence, provided that(a) at least three or four of the CDRs have no mutations and (b) theCDRs that do have mutations have only up to two or three amino acidsubstitutions, up to a contiguous five amino acid deletion, or acombination thereof In certain embodiments, nucleic acid moleculesencoding an antigen binding protein or antigen are used totransfect/transduce a host cell (e.g., hematopoietic stem cells, Tcells) for use in adoptive transfer therapy. Recent advances in methodsfor transfecting/transducing T-cells with desired nucleic acids havebeen described (e.g., US 2004/0087025), as have adoptive transferprocedures using T-cells of desired antigen-specificity (e.g., Schmittet al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742,2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther.18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011/0243972;US2011/0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), such thatadaptation of these methodologies to the presently disclosed embodimentsis contemplated, based on the teachings herein.

The antigen binding proteins or components as described herein may befunctionally characterized according to any of a large number of artaccepted methodologies for assaying T cell activity, includingdetermination of T cell binding, activation or induction and alsoincluding determination of T cell responses that are antigen-specific.Examples include determination of T cell proliferation, T cell cytokinerelease, antigen-specific T cell stimulation, MHC restricted T cellstimulation, CTL activity (e.g., by detecting ⁵¹Cr release frompre-loaded target cells), changes in T cell phenotypic markerexpression, and other measures of T-cell functions. Procedures forperforming these and similar assays are may be found, for example, inLefkovits (Immunology Methods Manual: The Comprehensive Sourcebook ofTechniques, 1998). See also Current Protocols in Immunology; Weir,Handbook of Experimental Immunology, Blackwell Scientific, Boston, Mass.(1986); Mishell and Shigii (eds.) Selected Methods in CellularImmunology, Freeman Publishing, San Francisco, Calif. (1979); Green andReed, Science 281:1309 (1998) and references cited therein).

Levels of cytokines may be determined according to methods describedherein and practiced in the art, including for example, ELISA, ELISPOT,intracellular cytokine staining, and flow cytometry and combinationsthereof (e.g., intracellular cytokine staining and flow cytometry).Immune cell proliferation and clonal expansion resulting from anantigen-specific elicitation or stimulation of an immune response may bedetermined by isolating lymphocytes, such as circulating lymphocytes insamples of peripheral blood cells or cells from lymph nodes, stimulatingthe cells with antigen, and measuring cytokine production, cellproliferation and/or cell viability, such as by incorporation oftritiated thymidine or non-radioactive assays, such as MTT assays andthe like. The effect of an immunogen described herein on the balancebetween a Th1 immune response and a Th2 immune response may be examined,for example, by determining levels of Th1 cytokines, such as IFN-γ,IL-12, IL-2, and TNF-β, and Type 2 cytokines, such as IL-4, IL-5, IL-9,IL-10, and IL-13.

Polynucleotides Encoding Antigen Binding Proteins or Antigens

Isolated or recombinant nucleic acid molecules encoding an antigenbinding protein like a CAR, high affinity recombinant TCR specific forantigen, or the antigen or an anti-idiotypic antibody against theantigen specific binding component as described herein may be producedand prepared according to various methods and techniques of themolecular biology or polypeptide purification arts. Construction of anexpression vector that is used for recombinantly producing an antigenbinding protein like a CAR, high affinity recombinant TCR specific forantigen, or the antigen or an anti-idiotypic antibody against theantigen specific binding component of interest can be accomplished byusing any suitable molecular biology engineering techniques known in theart, including the use of restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed in Sambrook et al. (1989 and 2001 editions; Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) andAusubel et al. (Current Protocols in Molecular Biology (2003)). Toobtain efficient transcription and translation, a polynucleotide in eachrecombinant expression construct includes at least one appropriateexpression control sequence (also called a regulatory sequence), such asa leader sequence and particularly a promoter operably (i.e.,operatively) linked to the nucleotide sequence encoding the immunogen.In certain embodiments, a polynucleotide is codon optimized forefficient expression in a target host cell.

Certain embodiments relate to nucleic acids that encode the polypeptidescontemplated herein, for instance, chimeric antigen receptors, highaffinity recombinant TCRs, the antigen of interest and anti-idiotypicantibodies specific for the antigen binding proteins. As one of skill inthe art will recognize, a nucleic acid may refer to a single- or adouble-stranded DNA, cDNA or RNA in any form, and may include a positiveand a negative strand of the nucleic acid which complement each other,including anti-sense DNA, cDNA and RNA. Also included are siRNA,microRNA, RNA—DNA hybrids, ribozymes, and other various naturallyoccurring or synthetic forms of DNA or RNA.

Standard techniques may be used for recombinant DNA, peptide andoligonucleotide synthesis, immunoassays and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques may be performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. These and related techniques and procedures may be generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references inmicrobiology, molecular biology, biochemistry, molecular genetics, cellbiology, virology and immunology techniques that are cited and discussedthroughout the present specification. See, e.g., Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (John Wiley and Sons, updated July 2008); ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I &II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols inImmunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H.Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY,NY); Real-Time PCR: Current Technology and Applications, Edited by JulieLogan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press,Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes,(Academic Press, New York, 1992); Guthrie and Fink, Guide to YeastGenetics and Molecular Biology (Academic Press, New York, 1991);Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, Eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R.Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCRProtocols (Methods in Molecular Biology) (Park, Ed., 3^(rd) Edition,2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998);Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and CC Blackwell, eds., 1986);Roitt, Essential Immunology, 6th Edition, (Blackwell ScientificPublications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols(Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); EmbryonicStem Cell Protocols: Volume I: Isolation and Characterization (Methodsin Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem CellProtocols: Volume II: Differentiation Models (Methods in MolecularBiology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem CellProtocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006);Mesenchymal Stem Cells: Methods and Protocols (Methods in MolecularBiology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. BunnellEds., 2008); Hematopoietic Stem Cell Protocols (Methods in MolecularMedicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001);Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (KevinD. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methodsin Molecular Biology) (Leslie P. Weiner Ed., 2008).

Certain embodiments include nucleic acids contained in a vector. One ofskill in the art can readily ascertain suitable vectors for use withcertain embodiments disclosed herein. A typical vector may comprise anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked, or which is capable of replication in a hostorganism. Some examples of vectors include plasmids, viral vectors,cosmids, and others. Some vectors may be capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors), whereas other vectors may be integrated into thegenome of a host cell upon introduction into the host cell and therebyreplicate along with the host genome. Additionally, some vectors arecapable of directing the expression of genes to which they areoperatively linked (these vectors may be referred to as “expressionvectors”). According to related embodiments, it is further understoodthat, if one or more agents (e.g., polynucleotides encoding an antigenbinding protein like a CAR, high affinity recombinant TCR specific forantigen, or the antigen or an anti-idiotypic antibody against theantigen specific binding component, as described herein) isco-administered to a subject, that each agent may reside in separate orthe same vectors, and multiple vectors (each containing a differentagent the same agent) may be introduced to a cell or cell population oradministered to a subject.

In certain embodiments, the nucleic acid encoding antigen bindingproteins or antigens of this disclosure may be operatively linked tocertain elements of a vector. For example, polynucleotide sequences thatare needed to effect the expression and processing of coding sequencesto which they are ligated may be operatively linked. Expression controlsequences may include appropriate transcription initiation, termination,promoter and enhancer sequences; efficient RNA processing signals suchas splicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequences); sequences that enhance protein stability;and possibly sequences that enhance protein secretion. Expressioncontrol sequences may be operatively linked if they are contiguous withthe gene of interest and expression control sequences that act in transor at a distance to control the gene of interest.

In particular embodiments, the recombinant expression vector isdelivered to an appropriate cell, for example, a hematopoietic stemcell, T cell, antigen-presenting cell (e.g., a dendritic cell) or thelike. The recombinant expression vectors may therefore also include, forexample, lymphoid tissue-specific transcriptional regulatory elements(TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specificTRE. Lymphoid tissue specific TRE are known in the art (see, e.g.,Thompson et al., Mol. Cell. Biol. 12:1043, 1992); Todd et al., J. Exp.Med. 177:1663, 1993); Penix et al., J. Exp. Med. 178:1483, 1993).

In addition to vectors, certain embodiments relate to host cells thatcomprise the vectors that are presently disclosed. One of skill in theart readily understands that many suitable host cells are available inthe art. A host cell may include any individual cell or cell culturewhich may receive a vector or the incorporation of nucleic acids and/orproteins, as well as any progeny cells. The term also encompassesprogeny of the host cell, whether genetically or phenotypically the sameor different. Suitable host cells may depend on the vector and mayinclude mammalian cells, animal cells, human cells, simian cells, insectcells, yeast cells, and bacterial cells. These cells may be induced toincorporate the vector or other material by use of a viral vector,transformation via calcium phosphate precipitation, DEAE-dextran,electroporation, microinjection, or other methods. For example, seeSambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (ColdSpring Harbor Laboratory, 1989).

Methods of Treatment

In certain aspects, the instant disclosure is directed to methods fortreating a hyperproliferative disorder or a condition (e.g.,characterized by antigen overexpression) by administering to humansubject an effective amount of a composition comprising a population ofmodified human T cells comprising a nucleic acid molecule that encodesan antigen binding protein, wherein the antigen binding proteincomprises a hydrophobic portion disposed between an extracellularbinding component and an intracellular effector component; and apopulation of modified human hematopoietic progenitor cells, modifiedhuman immune system cells or a combination thereof comprising a nucleicacid molecule that encodes the antigen specifically recognized by theextracellular binding component of the antigen binding protein. Incertain embodiments, the administration steps may be repeated multipletimes and for a period of a few weeks, a few months, or up to two yearsor more.

In certain embodiments, the hyperproliferative disorder is ahematological malignancy or a solid cancer. Exemplary hematologicalmalignancies include acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilicleukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma(NHL), or multiple myeloma (MM). Exemplary solid cancers include biliarycancer, bladder cancer, bone and soft tissue carcinoma, brain tumor,breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma,colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer,esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastomamultiforme, gynecological tumor, head and neck squamous cell carcinoma,hepatic cancer, lung cancer, malignant melanoma, osteosarcoma, ovariancancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primaryastrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer,renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissuesarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma,or uterine cancer.

In further aspects, the instant disclosure is directed to a for treatinga disease in a subject by (a) administering to a subject an effectiveamount of a population of modified human T cells comprising a nucleicacid molecule that encodes an antigen binding protein, wherein theantigen binding protein comprises a hydrophobic portion disposed betweenan extracellular binding component and an intracellular effectorcomponent; (b) administering to the subject an effective amount of apopulation of modified human hematopoietic progenitor cells, modifiedhuman immune system cells or a combination thereof comprising a nucleicacid molecule that encodes the antigen, wherein the extracellularbinding component of the antigen binding protein from the modified humanT cells of step (a) is specific for the antigen encoded by thepopulation of modified cells of this step (b); and (c) optionallyrepeating step (a), step (b) or both steps (a) and (b); thereby treatingdisease by adoptive cellular immunotherapy.

In still further aspects, the instant disclosure is directed to a methodfor improving adoptive cellular immunotherapy by (a) administering to asubject an effective amount of a population of modified human T cellscomprising a nucleic acid molecule that encodes an antigen bindingprotein, wherein the antigen binding protein comprises a hydrophobicportion disposed between an extracellular binding component and anintracellular effector component; and (b) administering to the subjectan effective amount of a population of modified human hematopoieticprogenitor cells, modified human immune system cells or a combinationthereof comprising a nucleic acid molecule that encodes the antigen,wherein the extracellular binding component of the antigen bindingprotein from the modified human T cells of step (a) is specific for theantigen encoded by the population of modified cells of this step (b).These administrations, which may be repeated as described herein,thereby boost, augment or enhance the efficacy of the adoptive cellularimmunotherapy.

In any of the aforementioned embodiments, the methods are used to treata viral disease, a bacterial disease, a cancer, an inflammatory disease,an immune disease, or an aging-associated disease.

In any of the aforementioned embodiments, the methods are used withcells encoding an antigen binding protein comprising a bindingcomponent, a hydrophobic portion and an intracellular effectorcomponent. For example the binding component may be an antibody variablefragment (Fv), a TCR variable domain, a receptor ectodomain, or aligand. In further embodiments, the binding component is a scFv or scTCRcomprising a variable region linker, such as a linker comprises a(Gly_(x)Ser_(y))_(n), wherein x and y are independently an integer from1 to 5, and n is an integer from 1 to 10. In further embodiments, thebinding component is specific for α-fetoprotein (AFP), B7H4, BTLA, CD3,CD19, CD20, CD25, CD22, CD28, CD30, CD40, CD44v6, CD52, CD56, CD79b,CD80, CD81, CD86, CD134 (0X40), CD137 (4-1BB), CD151, CD276, CA125, CEA,CEACAM6, c-Met, CT-7, CTLA-4, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4,EphA2, FLT1, FLT4, Frizzled, O-acetyl-GD2, GD2, GHRHR, GHR, GITR, gp130,HVEM, IGF1R, IL6R, KDR, L1CAM, Lewis A, Lewis Y, LTβR, LIFRβ, LRP5,MAGE, mesothelin, MUC1, NY-ESO-1, a cancer-specific neoantigen, OSMRβ,PD1, PD-L1, PD-L2, PSMA, PTCH1, RANK, Robol, ROR1, TERT, TGFBR2, TGFBR1,TLR7, TLR9, TNFRSF4, TNFR1, TNFR2, tyrosinase, TWEAK-R, or WT-1. In anyof these embodiments, the extracellular binding component of the antigenbinding protein from the modified human T cells is directed against adisease cell overexpressing the antigen.

In still further embodiments, the hydrophobic portion is a transmembranedomain, such as a CD4, CD8, CD28 or CD27 transmembrane domain. Incertain embodiments, an intracellular effector component comprises anintracellular region of CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD79A,CD79B, CD134, CD137, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS,Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk,SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combinationthereof. In particular embodiments, the intracellular effector componentcomprises CD3ζ and one or more of CD27, CD28, CD134, and CD137, or theintracellular effector component comprises LRP, NOTCH1, NOTCH2, NOTCH3,NOTCH4, ROR2, or Ryk.

In any of the aforementioned embodiments, the methods provide the use ofa modified immune system cell, such as a CD4+ T cell, a CD8+ T cell, aCD4− CD8− double negative T cell, a γδ T cell, regulatory T cell, anatural killer cell, a dendritic cell, or any combination thereof. Incertain embodiments, the T cell is a naïve T cell, a central memory Tcell, an effector memory T cell, or any combination thereof For example,the first population of modified T cells may consist essentially of CD4+T cells, CD8+ T cells, or both CD4+ and CD8+ T cells, and the secondpopulation of modified cells comprises modified human hematopoieticprogenitor cells. In other examples, the first population of modified Tcells may consist essentially of CD4+ T cells, CD8+ T cells, or bothCD4+ and CD8+ T cells, and the second population of modified cellscomprises modified human immune system cells, such as cells consistingessentially of CD4+ T cells, a CD8+ T cells, or both CD4+ and CD8+ Tcells.

In any of the aforementioned embodiments, the methods provide the use ofmodified cell populations that are recombinantly modified ex vivo via,for example, a viral vector, such as a lentiviral vector or aγ-retroviral vector. In further embodiments, the cell populations beingmodified are syngeneic, allogeneic, or autologous cells. In any of theaforementioned embodiments, the modified cell populations are furtherformulated with a pharmaceutically acceptable carrier, diluent, orexcipient as described herein.

In any of the aforementioned embodiments, the methods compriseadministering the modified cell populations intravenously. In any of theaforementioned embodiments, the methods comprise administering to thesubject a plurality of doses of the modified T cells from step (a), aplurality of doses of modified cells from step (b), or a combinationthereof. For example, the plurality of doses of modified T cells fromstep (a) are administered at intervals between administrations of aboutone week to about four weeks. In certain embodiments, the modified Tcells from step (a) are administered concurrently or sequentially withthe modified cells from step (b). In further embodiments, the initialdose of modified cells from step (b) are administered from about 1 dayto about 28 days after administering the modified T cells from step (a).

In any of the aforementioned embodiments, the methods compriseadministering the modified cells from step (b) sequentially within about24 hours of administering the modified T cells from step (a), or themodified T cells from step (a) sequentially within about 24 hours ofadministering the modified cells from step (b). In a specificembodiment, the initial administration comprises administering acomposition comprising a mixture of the modified T cells from step (a)and the modified cells from step (b), provided that the cells from step(b) are not activating the cells from step (a) until the mixture isadministered to a subject. In further embodiments, the modified cellsfrom step (b) are further administered in one or more doses at one ormore intervals for up to about 365 days after administering the modifiedT cells from step (a). In any of the aforementioned embodiments, themodified cells from step (b) comprise or consist essentially of modifiedhematopoietic progenitor cells, or comprise or consist essentially of Tcells.

In any of the aforementioned embodiments, the modified T cells from step(a) are administered to the subject at a dose of about 10⁶ cells/m² toabout 10¹¹ cells/m² and the modified T cells from step (b) areadministered to the subject at a dose of about 10⁶ cells/m² to about10¹¹ cells/m², wherein the doses for either or both modified cellpopulations may be repeated as described herein.

The presence of a hyperproliferative disorder or malignant condition ina subject refers to the presence of dysplastic, cancerous and/ortransformed cells in the subject, including, for example neoplastic,tumor, non-contact inhibited or oncogenically transformed cells, or thelike (e.g., solid cancers; hematologic cancers including lymphomas andleukemias, such as acute myeloid leukemia, chronic myeloid leukemia,etc.), which are known in the art and for which criteria for diagnosisand classification are established (e.g., Hanahan and Weinberg, 2011Cell 144:646; Hanahan and Weinberg 2000 Cell 100:57; Cavallo et al.,2011 Canc. Immunol. Immunother. 60:319; Kyrigideis et al., 2010 J.Carcinog. 9:3). In certain embodiments, such cancer cells may be cellsof acute myeloid leukemia, B-cell lymphoblastic leukemia, T-celllymphoblastic leukemia, or myeloma, including cancer stem cells that arecapable of initiating and serially transplanting any of these types ofcancer (see, e.g., Park et al., Molec. Therap. 17:219, 2009).

In another aspect, the present disclosure provides a method forinhibiting growth, metastasis or metastatic growth of a malignancy(e.g., a solid malignancy or a hematologic malignancy), comprisingadministering to a subject in need thereof an effective amount of a cellencoding a polypeptide complex provided herein or a composition thereof.

A wide variety of cancers, including solid malignancy and hematologicmalignancy, are amenable to the compositions and methods disclosedherein. Types of cancer that may be treated include adenocarcinoma ofthe breast, prostate, pancreas, colon and rectum; all forms ofbronchogenic carcinoma of the lung (including squamous cell carcinoma,adenocarcinoma, small cell lung cancer and non-small cell lung cancer);myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma;choristoma; branchioma; malignant carcinoid syndrome; carcinoid heartdisease; and carcinoma (e.g., Walker, basal cell, basosquamous,Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous,non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,bronchogenic, squamous cell, and transitional cell). Additional types ofcancers that may be treated include: histiocytic disorders; leukemia;histiocytosis malignant; Hodgkin's disease; non-Hodgkin's lymphoma;plasmacytoma; reticuloendotheliosis; melanoma; renal cell carcinoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastictumor.

Further, the following types of cancers are also contemplated asamenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma;cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma;sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma;myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma;ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma;neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma;paraganglioma; paraganglioma nonchromaffin; and glioblastoma multiforme.The types of cancers that may be treated also include angiokeratoma;angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma;lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma;leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma;ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms;nerofibromatosis; and cervical dysplasia.

Additional exemplary cancers that are also amenable to the compositionsand methods disclosed herein are B-cell cancers, including B-celllymphomas [such as various forms of Hodgkin's disease, non-Hodgkin'slymphoma (NHL) or central nervous system lymphomas], leukemias [such asacute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),Hairy cell leukemia and chronic myoblastic leukemia] and myelomas (suchas multiple myeloma). Additional B cell cancers include smalllymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacyticlymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitaryplasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginalzone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodalmarginal zone B-cell lymphoma, follicular lymphoma, mantle celllymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) largeB-cell lymphoma, intravascular large B-cell lymphoma, primary effusionlymphoma, Burkitt lymphoma/leukemia, B-cell proliferations of uncertainmalignant potential, lymphomatoid granulomatosis, and post-transplantlymphoproliferative disorder.

In certain embodiments, cells encoding antigen binding proteins usefulfor inhibiting growth of a solid malignancy or metastasis or metastaticgrowth of a solid malignancy or a hematologic malignancy include thosethat specifically bind to a tumor or cancer antigen and a second targetantigen on the cancer cell.

As understood by a person skilled in the medical art, the terms, “treat”and “treatment,” refer to medical management of a disease, disorder, orcondition of a subject (i.e., patient, host, who may be a human ornon-human animal) (see, e.g., Stedman's Medical Dictionary). In general,an appropriate dose and treatment regimen provide one or more of anantigen binding protein (e.g., CAR) or high affinity recombinant TCRspecific for a human target antigen a host T cell expressing the same,and optionally an adjunctive therapy (e.g., a cytokine such as IL-2,IL-15, IL-21 or any combination thereof), in an amount sufficient toprovide therapeutic or prophylactic benefit. Therapeutic or prophylacticbenefit resulting from therapeutic treatment or prophylactic orpreventative methods include, for example an improved clinical outcome,wherein the object is to prevent or retard or otherwise reduce (e.g.,decrease in a statistically significant manner relative to an untreatedcontrol) an undesired physiological change or disorder, or to prevent,retard or otherwise reduce the expansion or severity of such a diseaseor disorder. Beneficial or desired clinical results from treating asubject include abatement, lessening, or alleviation of symptoms thatresult from or are associated the disease or disorder to be treated;decreased occurrence of symptoms; improved quality of life; longerdisease-free status (i.e., decreasing the likelihood or the propensitythat a subject will present symptoms on the basis of which a diagnosisof a disease is made); diminishment of extent of disease; stabilized(i.e., not worsening) state of disease; delay or slowing of diseaseprogression; amelioration or palliation of the disease state; andremission (whether partial or total), whether detectable orundetectable; or overall survival.

“Treatment” can also mean prolonging survival when compared to expectedsurvival if a subject were not receiving treatment. Subjects in need ofthe methods and compositions described herein include those who alreadyhave the disease or disorder, as well as subjects prone to have or atrisk of developing the disease or disorder. Subjects in need ofprophylactic treatment include subjects in whom the disease, condition,or disorder is to be prevented (i.e., decreasing the likelihood ofoccurrence or recurrence of the disease or disorder). The clinicalbenefit provided by the compositions (and preparations comprising thecompositions) and methods described herein can be evaluated by designand execution of in vitro assays, preclinical studies, and clinicalstudies in subjects to whom administration of the compositions isintended to benefit, as described in the examples.

Cells expressing an antigen binding protein or high affinity recombinantTCR specific for human disease-associated antigen, along with cellsexpressing the target antigen, as described herein, may be administeredto a subject in a pharmaceutically or physiologically acceptable orsuitable excipient or carrier. Pharmaceutically acceptable excipientsare biologically compatible vehicles, e.g., physiological saline, whichare described in greater detail herein, that are suitable foradministration to a human or other non-human mammalian subject.

A therapeutically effective dose is an amount of host T cells(expressing an antigen binding protein or high affinity recombinant TCRspecific for a human disease-associated antigen) and cells expressingthe target antigen used in adoptive transfer that is capable ofproducing a clinically desirable result (i.e., a sufficient amount toinduce or enhance a specific T cell immune response against cellsoverexpressing antigen (e.g., a cytotoxic T cell response) in astatistically significant manner) in a treated human or non-humanmammal. As is well known in the medical arts, the dosage for any onepatient depends upon many factors, including the patient's size, weight,body surface area, age, the particular therapy to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Doses will vary, but a preferred dose foradministration of a host cell comprising a recombinant expression vectoras described herein is about 10⁶ cells/m², about 5×10⁶ cells/m², about10⁷ cells/m², about 5×10⁷ cells/m², about 10⁸ cells/m², about 5×10⁸cells/m ², about 10⁹ cells/m ², about 5×10⁹ cells/m², about 10¹⁰cells/m², about 5×10¹⁰ cells/m², or about 10¹¹ cells/m².

Pharmaceutical compositions may be administered in a manner appropriateto the disease or condition to be treated (or prevented) as determinedby persons skilled in the medical art. An appropriate dose and asuitable duration and frequency of administration of the compositionswill be determined by such factors as the health condition of thepatient, size of the patient (i.e., weight, mass, or body area), thetype and severity of the patient's disease, the particular form of theactive ingredient, and the method of administration. In general, anappropriate dose and treatment regimen provide the composition(s) in anamount sufficient to provide therapeutic and/or prophylactic benefit(such as described herein, including an improved clinical outcome, suchas more frequent complete or partial remissions, or longer disease-freeand/or overall survival, or a lessening of symptom severity). Forprophylactic use, a dose should be sufficient to prevent, delay theonset of, or diminish the severity of a disease associated with diseaseor disorder. Prophylactic benefit of the immunogenic compositionsadministered according to the methods described herein can be determinedby performing pre-clinical (including in vitro and in vivo animalstudies) and clinical studies and analyzing data obtained therefrom byappropriate statistical, biological, and clinical methods andtechniques, all of which can readily be practiced by a person skilled inthe art.

A condition associated with antigen overexpression includes any disorderor condition in which underactivity, overactivity or improper activityof an antigen-associated cellular or molecular event is present, andtypically results from unusually high (with statistical significance)levels of antigen expression in afflicted cells (e.g., leukemic cells),relative to normal cells. A subject having such a disorder or conditionwould benefit from treatment with a composition or method of thepresently described embodiments. Some conditions associated with antigenoverexpression thus may include acute as well as chronic disorders anddiseases, such as those pathological conditions that predispose thesubject to a particular disorder.

Some examples of conditions associated with antigen overexpressioninclude hyperproliferative disorders, which refer to states of activatedand/or proliferating cells (which may also be transcriptionallyoveractive) in a subject including tumors, neoplasms, cancer,malignancy, etc. In addition to activated or proliferating cells, thehyperproliferative disorder may also include an aberration ordysregulation of cell death processes, whether by necrosis or apoptosis.Such aberration of cell death processes may be associated with a varietyof conditions, including cancer (including primary, secondarymalignancies as well as metastasis), or other conditions.

According to certain embodiments, virtually any type of cancer that ischaracterized by antigen expression or overexpression may be treatedthrough the use of compositions and methods disclosed herein, includinghematological cancers (e.g., leukemia including acute myeloid leukemia(AML), T or B cell lymphomas, myeloma, and others). Furthermore,“cancer” may refer to any accelerated proliferation of cells, includingsolid tumors, ascites tumors, blood or lymph or other malignancies;connective tissue malignancies; metastatic disease; minimal residualdisease following transplantation of organs or stem cells; multi-drugresistant cancers, primary or secondary malignancies, angiogenesisrelated to malignancy, or other forms of cancer. Also contemplatedwithin the presently disclosed embodiments are specific embodimentswherein only one of the above types of disease is included, or wherespecific conditions may be excluded regardless of whether or not theyare characterized by target antigen overexpression.

Certain methods of treatment or prevention contemplated herein includeadministering a host cell (which may be autologous, allogeneic orsyngeneic) comprising a desired nucleic acid molecule as describedherein that is stably integrated into the chromosome of the cell. Forexample, such a cellular composition may be generated ex vivo usingautologous, allogeneic or syngeneic immune system cells (e.g., T cells,antigen-presenting cells, natural killer cells) in order to administer adesired, antigen-targeted T-cell composition to a subject, along with anantigen expressing cell composition as an adoptive immunotherapy.

As used herein, administration of a composition or therapy refers todelivering the same to a subject, regardless of the route or mode ofdelivery. Administration may be effected continuously or intermittently,and parenterally. Administration may be for treating a subject alreadyconfirmed as having a recognized condition, disease or disease state, orfor treating a subject susceptible to or at risk of developing such acondition, disease or disease state. Co-administration with anadjunctive therapy may include simultaneous and/or sequential deliveryof multiple agents in any order and on any dosing schedule (e.g.,antigen specific recombinant host T cells and antigen expressing cellswith one or more cytokines; immunosuppressive therapy such ascalcineurin inhibitors, corticosteroids, microtubule inhibitors, lowdose of a mycophenolic acid prodrug, or any combination thereof).

In certain embodiments, a plurality of doses of a recombinant host Tcell as described herein is administered to the subject, which may beadministered at intervals between administrations of about two to aboutfour weeks, and the subject may be optionally administered an antigenexpressing cell as described herein simultaneously, concurrently orsubsequent to the administrations of the recombinant (modified) T cells.In further embodiments, a cytokine is administered sequentially,provided that the subject was administered the recombinant host T cellat least three or four times before cytokine administration. In certainembodiments, a cytokine is administered subcutaneously (e.g., IL-2,IL-15, IL-21). In still further embodiments, the subject being treatedis further receiving immunosuppressive therapy, such as calcineurininhibitors, corticosteroids, microtubule inhibitors, low dose of amycophenolic acid prodrug, or any combination thereof. In yet furtherembodiments, a subject being treated has received a non-myeloablative ora myeloablative hematopoietic cell transplant (e.g., autologous,allogeneic), wherein the treatment may be administered at least two toat least three months after the myeloablative or non-myeloablativehematopoietic cell transplant.

An effective amount of a therapeutic or pharmaceutical compositionrefers to an amount sufficient, at dosages and for periods of timeneeded, to achieve the desired clinical results or beneficial treatment,as described herein. An effective amount may be delivered in one or moreadministrations. If the administration is to a subject already known orconfirmed to have a disease or disease-state, the term “therapeuticamount” may be used in reference to treatment, whereas “prophylacticallyeffective amount” may be used to describe administrating an effectiveamount to a subject that is susceptible or at risk of developing adisease or disease-state (e.g., recurrence) as a preventative course.

The level of a CAR-CTL immune response may be determined by any one ofnumerous immunological methods described herein and routinely practicedin the art. The level of a CAR-CTL immune response may be determinedprior to and following administration of any one of the herein describedmodified hematopoietic stem cell, immune system cell (e.g., T cell) orany combination thereof. Cytotoxicity assays for determining CTLactivity may be performed using any one of several techniques andmethods routinely practiced in the art (see, e.g., Henkart et al.,“Cytotoxic T-Lymphocytes” in Fundamental Immunology, Paul (ed.) (2003Lippincott Williams & Wilkins, Philadelphia, Pa.), pages 1127-50, andreferences cited therein). Antigen-specific T cell responses aretypically determined by comparisons of observed T cell responsesaccording to any of the herein described T cell functional parameters(e.g., proliferation, cytokine release, CTL activity, altered cellsurface marker phenotype, etc.) that may be made between T cells thatare exposed to a cognate antigen in an appropriate context (e.g., theantigen or Ag-APC used to prime or activate the modified T cells) and Tcells from the same source population that are exposed instead to astructurally distinct or irrelevant control antigen. A response to thecognate antigen that is greater, with statistical significance, than theresponse to the control antigen signifies antigen-specificity.

A biological sample may be obtained from a subject for determining thepresence and level of an immune response as described herein. A“biological sample” as used herein may be a blood sample (from whichserum or plasma may be prepared), biopsy specimen, body fluids (e.g.,lung lavage, ascites, mucosal washings, synovial fluid), bone marrow,lymph nodes, tissue explant, organ culture, or any other tissue or cellpreparation from the subject or a biological source. Biological samplesmay also be obtained from the subject prior to receiving any immunogeniccomposition, which biological sample is useful as a control forestablishing baseline (i.e., pre-immunotherapy) data.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers may be frozen to preserve the stability of theformulation until. In certain embodiments, a unit dose comprises arecombinant host cell as described herein at a dose of about 10⁶cells/m² to about 10¹¹ cells/m². The development of suitable dosing andtreatment regimens for using the particular compositions describedherein in a variety of treatment regimens, including e.g., parenteral orintravenous administration or formulation.

If the subject composition is administered parenterally, the compositionmay also include sterile aqueous or oleaginous solution or suspension.Suitable non-toxic parenterally acceptable diluents or solvents includewater, Ringer's solution, isotonic salt solution, 1,3-butanediol,ethanol, propylene glycol or polythethylene glycols in mixtures withwater. Aqueous solutions or suspensions may further comprise one or morebuffering agents, such as sodium acetate, sodium citrate, sodium borateor sodium tartrate. Of course, any material used in preparing any dosageunit formulation should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.Dosage unit form, as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unit maycontain a predetermined quantity of recombinant cells or active compoundcalculated to produce the desired therapeutic effect in association withan appropriate pharmaceutical carrier.

In general, an appropriate dosage and treatment regimen provides theactive molecules or cells in an amount sufficient to provide therapeuticor prophylactic benefit. Such a response can be monitored byestablishing an improved clinical outcome (e.g., more frequentremissions, complete or partial, or longer disease-free survival) intreated subjects as compared to non-treated subjects. Increases inpreexisting immune responses to a tumor protein generally correlate withan improved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which are routine in the art and may be performed using samples obtainedfrom a subject before and after treatment.

EXAMPLES Example 1 Characterization of ROR1 CAR-T Cells for AdoptiveTransfer Transfer Immunotherapy

Human and macaque 4-1BB and CD3ζ signaling molecules are highlyconserved (95%), and there is 100% identity of the immunoreceptortyrosine-based activation motifs of CD3ζ. To ensure the ROR1⁺ CARs werefunctional in macaque T cells, T cells were transduced with ROR1 CARsencoding human or macaque 4-1BB and CD3ζ signaling domains, andrecognition of ROR1⁺ target cells was evaluated. To provide a selectionmarker to purify transduced T cells, a sequence encoding rhesus tCD19was incorporated in the vector downstream of a T2A ribosomal skipelement (Berger et al., J. Med. Primatol. 40:88, 2011). The recognitionof K562/ROR1 tumor cells by macaque T cells transduced with either aROR1 CAR containing human 4-1BB /CD3ζ or a ROR1 CAR containing macaque4-1BB/CD3ζ was equivalent (FIG. 1A). Accordingly, in vivo safety studiesusing the construct containing the human signaling domains were used toexamine the potential clinical translation of this vector.

For adoptive transfer experiments, samples of peripheral blood wereobtained from three adult rhesus macaques, and PBMC from each animalwere stimulated with anti-CD3/CD28 mAbs, and then the T cells weretransduced with a ROR1 CAR retroviral vector. The mean transductionefficiency of CD4⁺ and CD8⁺ T cells as measured by co-expression oftCD19 was 28.2% (6.6-58.1%) and the transduced cells were enriched togreater than 90% purity by immuno-magnetic selection 4-5 days aftertransduction (FIG. 1B). Following expansion, a second immuno-magneticselection was performed to purify CD4⁺ and CD8⁺ CAR-T cells, and eachsubset was expanded separately to allow formulation of an a 1:1 mixture.This ensured a uniform composition of ROR1 CAR-T cells in each adoptivetransfer experiment. In addition, autologous CD4⁺ and CD8⁺ T cells thatwere modified to express EGFRt or tCD34 were generated, and prepared asa 1:1 mixture of control transduced CD4⁺ and CD8⁺ T cells forco-infusion with ROR1 CAR-T cells.

The function of the ROR1 CAR in CD4⁺ and CD8⁺ T cells was confirmed bycytotoxicity, proliferation, and cytokine-release assays afterco-culture with K562/ROR1 cells. CD8⁺ ROR1 CAR-T cells lysed K562/ROR1targets more efficiently than CD4⁺ ROR1 CAR-T cells (FIG. 1C), and bothsubsets proliferated and produced cytokines specifically in response toK562/ROR1 cells (FIG. 1D).

Example 2 ROR1 CAR-T Cells are Functional In Vivo

To determine if autologous ROR1 CAR-T cells could be transferred safelyand to analyze their persistence and migration in vivo, a dose of 1×10⁸ROR1 CAR-T cells/kg (CD4:CD8 ratio of 1:1) were administered to a singlemacaque. This cell dose is less than the dose of CMV-specific T cellsadministered safely to macaques but equals or exceeds the dose used inclinical trials of CAR-T cell therapy for CD19⁺ B cell malignancies(Maus et al., Blood 123:2625, 2014). Concurrently, the same dose ofEGFRt⁺ T cells was administered to control for cell persistence andmigration. The animal was monitored after the T cell infusions forfever, respiratory distress, appetite, diarrhea, and weight loss, andexamined pre- and post-infusion blood samples for CBC, serum chemistry,and cytokine levels. No immediate or delayed clinical abnormalities wereobserved at this cell dose. Body weight, CBC, and serum chemistryremained within normal limits, apart from a transient increase in themuscle-derived creatine phosphokinase (CPK) observed in macaques afterintra-muscular (i.m.) ketamine injections for sedation of the animalprior to T-cell infusions (FIG. 2A). Plasma levels of IFN-γ and IL-6were increased on day 1 after ROR1 CAR-T cells, but returned to normalby day 3 (FIG. 2B). Increases in IFN-γ and IL-6 were not observed aftertransferring a 5-times higher cell dose of autologous T cells transducedwith only a marker gene to a separate animal (data not shown),indicating that the elevated cytokine levels after infusing ROR1 CAR-Tcells reflected transient activation of the CAR-T cells in vivo.

Analysis of the frequency of ROR1 CAR-T cells and control T cells in theblood revealed a difference beginning at the earliest time points afterinfusion. ROR1 CAR-T cells were detected in the blood one day after theinfusion at a frequency of 0.3% of CD4⁺ and 0.8% of CD8⁺ T cells (7cells/μL and 6 cells/μL), and persisted at levels of 4-11 cells/4 overthe following 2 weeks. The frequencies of control CD4⁺ and CD8⁺ EGFRe Tcells were higher on day 1 (6.2% of CD4⁺ and 4.6% of CD8⁺ T cellscorresponding to 137 cells/4 and 36 cells/4). The EGFRe T cellsgradually declined to stable levels of 6-13 cells/4 during the 4-weekfollow up (FIG. 2C-D). qPCR analysis for transgene-specific sequencesconfirmed the distinct pattern of in vivo persistence (FIG. 2E).

The persistence data indicated that the ROR1 CAR-T cells may bemigrating from the blood, perhaps into tissues where ROR1 might beexpressed. It was previously shown that a subset of immature B cells inthe bone marrow (BM) expresses ROR1 and adoptively transferred macaque Tcells migrate to BM and lymph nodes (LNs) (Hudecek et al., Blood116:4532, 2010). Therefore, aliquots of BM and LN samples obtained priorto and on day 5 after infusion was examined for the presence of bothROR1⁺ B cells and the transferred T cells. EGFRt⁺ and ROR1 CAR-T cellswere present in the day 5 post-infusion BM and LN samples, but the ROR1CAR-T cells were present at 1.1-2.3-fold higher frequency than thecontrol EGFRt⁺ T cells (FIG. 3A). A gating strategy to detect the subsetof CD19⁺CD45^(intermediate) B cells was used on BM cell suspensions,which contain the pre-BII-large stage of B-cells that expresses ROR1 inhumans (data not shown). ROR1 expression was detected on a subset ofCD19⁺CD45^(intermediate) B cells (8.3%) in the BM obtained prior to theT-cell infusion, and this subset was eliminated in the BM sampleobtained on day 5 after transfer of ROR1 CAR-T cells (FIG. 3B).Enumeration of the peripheral blood CD19⁺ B cells, which are ROR1⁻,demonstrated that the ROR1 CAR-T cells had no effect on the mature CD19⁺B cell pool over the 28 days of the study (FIG. 3C). These resultsdemonstrate that transferred ROR1 CAR-T cells migrated to the BM andrecognized and eliminated ROR1⁺ B-cell precursors, but did not causeorgan toxicity or deplete circulating mature B cells.

To confirm that the ROR1 CAR-T cells remained functional in vivo, PBMCobtained one week post-infusion were stimulated with K562/ROR1 cells,PMA/Ionomycin, or media alone. Pre-infusion PBMC and ROR1 CAR-T cellsserved as controls in the assay. After 4 hours of stimulation, the cellswere analyzed for expression of CD107A, which is a marker ofdegranulation after antigen recognition by CD8⁺ T cells (Chan and Kaur,J. Immunol. Methods 325:20, 2007). An increase in CD107A expression wasdetected in the subset of CD8⁺ ROR1 CAR-T cells persisting in vivo afterstimulation, and this increase was at a similar level as that observedin ROR1 CAR-T cells prior to infusion (FIG. 3D). Thus, transferred ROR1CAR-T cells that persisted in the blood in vivo remained functionalindicating the lack of observed organ toxicity beyond depletion ofB-cell precursors was not due to dysfunction of ROR1 CAR-T cells.

EXAMPLE 3 ROR1 CAR-T Cells Respond to Antigen In Vivo

The infusion of higher CAR-T cell doses may be necessary to treat ROR1⁺malignancies and might reveal toxicities to normal tissues, particularlyif the CAR-T cells were activated in vivo by recognition of tumor cellsexpressing high levels of ROR1. During analysis of ROR1-expression on Bcells in macaques, it was observed that unlike humans, some animals havea high frequency of mature B cells in the LNs that express ROR1 (datanot shown). Two animals with high levels of ROR1⁺ B cells in LN wereselected for analysis of the safety of a higher dose (5×10⁸/kg) of ROR1CAR-T cells since experience in the first animal suggested thatreduction of ROR1⁺ B cells provided a surrogate for in vivo function ofCAR-T cells. A tCD34 was used as a marker gene in the control T cells inthese animals since CD34-enrichment was more efficient thanEGFRt-selection, which facilitated obtaining the higher T-cell doseneeded for this experiment. To maximize the possibility that activatingROR1 CAR-T cells in vivo might reveal toxicity, an infusion ofautologous T cells transfected to express cell-surface tROR1 five daysafter administration of CAR-T cells was used if no toxicity was observedpreviously (FIG. 4A).

The higher dose of ROR1 CAR-T cells were well tolerated and althoughCD4⁺ and CD8⁺ ROR1 CAR-T cells were readily detectable in both animals,a reduced frequency of ROR1 CAR-T cells was again observed as comparedto control tCD34 T cells in the blood one day after adoptive transfer(FIG. 4B). The difference in the peak frequency did not reflect uniqueproperties of the marker genes that might influence migration sinceco-infusion of T cells marked only with tCD19 or tCD34 in a controlanimal provided equivalently high levels of both populations in vivo(data not shown).

Cell suspensions from PBMC, BM and LNs obtained from each animal at day3 after infusion were stained for the presence of the transferred Tcells. ROR1 CAR-T cells, but not control tCD34 ⁺ T cells were present ata higher frequency in the BM than in the PBMC in both animals, and thefrequency of ROR1 CAR-T cells in the LN exceeded that of control tCD34 ⁺T cells (FIG. 4C). The accumulation of ROR1 CAR-T cells in BM and LN atday 3 after adoptive transfer coincided with a >65%-80% reduction ofROR1⁺ B cells compared to the pre-treatment BM and LNs (see FIGS. 4D and4E). These data are consistent with that observed in the first animaltreated with a lower dose of ROR1 CAR-T cells and shows that transferredROR1 CAR-T cells migrate out of blood and accumulate at sites whereROR1⁺ B cells reside.

To determine if systemically activating the ROR1 CAR-T cells in vivomight reveal toxicity, autologous T cells that were transduced toexpress tROR1 (tROR⁺ T cells) were infused into each animal. In vitro,tROR1-modified T cells were recognized as efficiently as K562/ROR1 cellsby autologous ROR1 CAR-T cells (data not shown). The infusion of tROR⁺ Tcells did not cause acute side effects in either animal, and increasesin CD4⁺ and CD8⁺ ROR1 CAR-T cells was observed to 30 cells/μL and 52cells/μL (A13011), and to 22 cells/4 and 34 cells/μL (A13002),respectively, over 5-7 days (FIG. 5A). This represented an up to7.7-fold (A13011) and 4.3-fold (A13002) increase in ROR1 CAR-T cellswithout substantial changes in numbers of endogenous or tCD34-markedCD4⁺ and CD8⁺ T cells (FIG. 5A-B). To determine if the ROR1 CAR-T cellseliminated the tROR⁺ T cells in vivo, samples of PBMC obtained on day 1,5, and 7 after T-APC challenge were examined for the presence ofcirculating tROR⁺ T cells. In A13011, the tROR⁺ T cells were present ata frequency of 1.0% of CD8⁺ T cells one day after transfer and themajority of these cells displayed only low-level ROR1-expressioncompared to the input tROR1⁺ T cells (FIG. 5C). Importantly, theROR1^(low) T cells declined further to a frequency of 0.3% and <0.1% ofCD8³⁰ T cells by day 5 and 7, respectively (FIG. 5C). A similar patternof tROR⁺ T cell persistence was observed in A13002, with only rareROR1^(low) T cells persisting in vivo (data not shown). By contrast, thefrequencies of tROR1⁺ T cells in the blood at days 1, 5 and 7 were 5.5%,8.6%, and 8.0% after infusion of these T cells to an animal withoutdetectable ROR1 CAR-T cells (FIGS. 5C and 5D).

In sum, this demonstrates that recombinantly created “antigen presentingcells” (in this case T cells producing antigen, but other immune systemcells and even hematopoietic stem cells may be used as an APC)expressing the same antigen being recognized by a CAR modified T cellcan boost or enhance an adoptive transfer immunotherapy.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. A method for improving adoptive cellularimmunotherapy, comprising: (a) administering to a subject an effectiveamount of a population of modified human T cells comprising a nucleicacid molecule that encodes an antigen binding protein, wherein theantigen binding protein comprises a hydrophobic portion disposed betweenan extracellular binding component and an intracellular effectorcomponent; and (b) administering to the subject an effective amount of apopulation of modified human hematopoietic progenitor cells, modifiedhuman immune system cells or a combination thereof comprising a nucleicacid molecule that encodes the antigen, wherein the extracellularbinding component of the antigen binding protein from the modified humanT cells of step (a) is specific for the antigen encoded by thepopulation of modified cells of this step (b); thereby boosting,augmenting or enhancing the efficacy of the adoptive cellularimmunotherapy.
 2. A method for treating a disease in a subject,comprising: (a) administering to a subject an effective amount of apopulation of modified human T cells comprising a nucleic acid moleculethat encodes an antigen binding protein, wherein the antigen bindingprotein comprises a hydrophobic portion disposed between anextracellular binding component and an intracellular effector component;(b) administering to the subject an effective amount of a population ofmodified human hematopoietic progenitor cells, modified human immunesystem cells or a combination thereof comprising a nucleic acid moleculethat encodes the antigen, wherein the extracellular binding component ofthe antigen binding protein from the modified human T cells of step (a)is specific for the antigen encoded by the population of modified cellsof this step (b); and (c) optionally repeating step (a), step (b) orboth steps (a) and (b); thereby treating disease by adoptive cellularimmunotherapy.
 3. The method according to claim 2, wherein the diseaseis a viral disease, a bacterial disease, a cancer, an inflammatorydisease, an immune disease, or an aging-associated disease.
 4. A methodfor treating a hyperproliferative disease, comprising: (a) administeringto a subject an effective amount of a population of modified human Tcells comprising a nucleic acid molecule that encodes an antigen bindingprotein, wherein the antigen binding protein comprises a hydrophobicportion disposed between an extracellular binding component and anintracellular effector component; (b) administering to the subject aneffective amount of a population of modified human hematopoieticprogenitor cells, modified human immune system cells or a combinationthereof comprising a nucleic acid molecule that encodes the antigen,wherein the extracellular binding component of the antigen bindingprotein from the modified human T cells of step (a) is specific for theantigen encoded by the population of modified cells of this step (b);and (c) optionally repeating step (a), step (b) or both steps (a) and(b); thereby treating the hyperproliferative disease.
 5. The methodaccording to claim 4, wherein the hyperproliferative disorder is ahematological malignancy or a solid cancer.
 6. The method according toclaim 5, wherein the hematological malignancy is selected from acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronicmyelogenous leukemia (CML), chronic eosinophilic leukemia (CEL),myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), ormultiple myeloma (MM).
 7. The method according to claim 5, wherein thesolid cancer is selected from biliary cancer, bladder cancer, bone andsoft tissue carcinoma, brain tumor, breast cancer, cervical cancer,colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoidtumor, embryonal cancer, endometrial cancer, esophageal cancer, gastriccancer, gastric adenocarcinoma, glioblastoma multiforme, gynecologicaltumor, head and neck squamous cell carcinoma, hepatic cancer, lungcancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreaticcancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor,primary thyroid cancer, prostate cancer, renal cancer, renal cellcarcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma,testicular germ-cell tumor, urothelial cancer, uterine sarcoma, oruterine cancer.
 8. The method according to any one of claims 1-7,wherein the binding component is an antibody variable fragment (Fv), aTCR variable domain, a receptor ectodomain, or a ligand.
 9. The methodaccording to claim 8, wherein the binding component is a scFv or scTCRcomprising a variable region linker.
 10. The method according to claim9, wherein the variable region linker comprises a (Gly_(x)Ser_(y))_(n),wherein x and y are independently an integer from 1 to 5, and n is aninteger from 1 to
 10. 11. The method according to any one of claims1-10, wherein the hydrophobic portion is a transmembrane domain.
 12. Themethod according to claim 11, wherein the transmembrane domain is a CD4,CD8, CD28 or CD27 transmembrane domain.
 13. The method according to anyone of claims 1-12, wherein the intracellular effector componentcomprises an intracellular region of CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28,CD79A, CD79B, CD134, CD137, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM,ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2,Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or anycombination thereof.
 14. The method according to any one of claims 1-13,wherein the intracellular effector component comprises CD3ζ and one ormore of CD27, CD28, CD134, and CD137.
 15. The method according to anyone of claims 1-13, wherein the intracellular effector componentcomprises LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, or Ryk.
 16. Themethod according to any one of claims 1-15, wherein the bindingcomponent is specific for α-fetoprotein (AFP), B7H4, BTLA, CD3, CD19,CD20, CD25, CD22, CD28, CD30, CD40, CD44v6, CD52, CD56, CD79b, CD80,CD81, CD86, CD134 (OX40), CD137 (4-1BB), CD151, CD276, CA125, CEA,CEACAM6, c-Met, CT-7, CTLA-4, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4,EphA2, FLT1, FLT4, Frizzled, O-acetyl-GD2, GD2, GHRHR, GHR, GITR, gp130,HVEM, IGF1R, IL6R, KDR, L1CAM, Lewis A, Lewis Y, LTβR, LIFRβ, LRP5,MAGE, mesothelin, MUC1, NY-ESO-1, a cancer-specific neoantigen, OSMRβ,PD1, PD-L1, PD-L2, PSMA, PTCH1, RANK, Robo1, ROR1, TERT, TGFBR2, TGFBR1,TLR7, TLR9, TNFRSF4, TNFR1, TNFR2, tyrosinase, TWEAK-R, or WT-1.
 17. Themethod according to any one of claims 1-16, wherein the antigen bindingprotein is a T cell receptor (TCR) or a chimeric antigen receptor. 18.The method according to any one of claims 1-17, wherein the immunesystem cell is a CD4− T cell, a CD8+ T cell, a CD4− CD8− double negativeT cell, a γδ T cell, a natural killer cell, a dendritic cell, or anycombination thereof.
 19. The method according to any one of claims 1-18,wherein the modified T cell is a naïve T cell, a central memory T cell,an effector memory T cell, or any combination thereof.
 20. The methodaccording to any one of claims 1-19, wherein the first population ofmodified T cells consists essentially of CD4+ T cells, a CD8+ T cells,or both CD4+ and CD8+ T cells.
 21. The method according to any one ofclaims 1-20, wherein the second population of modified cells comprisesmodified human hematopoietic progenitor cells.
 22. The method accordingto any one of claims 1-20, wherein the second population of modifiedcells comprises modified human immune system cells.
 23. The methodaccording to claim 22, wherein the modified immune system cells consistessentially of CD4+ T cells, a CD8+ T cells, or both CD4+ and CD8+ Tcells.
 24. The method according to any one of claims 1-23, wherein thecells are recombinantly modified ex vivo.
 25. The method according toclaim 24, wherein the ex vivo modified cells are modified using a viralvector.
 26. The method according to claim 25, wherein the viral vectoris a lentiviral vector or a γ-retroviral vector.
 27. The methodaccording to any one of claims 1-26, wherein the population of modifiedcells is allogeneic, syngeneic, or autologous.
 28. The method accordingto any one of claims 1-27, wherein the extracellular binding componentof the antigen binding protein from the modified human T cells isdirected against a cell overexpressing the antigen.
 29. The methodaccording to any one of claims 1-28, wherein the modified cells areadministered intravenously.
 30. The method according to any one ofclaims 1-29, wherein the method comprises administering to the subject aplurality of doses of the modified T cells from step (a), a plurality ofdoses of modified cells from step (b), or a combination thereof.
 31. Themethod according to claim 30, wherein the plurality of doses of modifiedT cells from step (a) are administered at intervals betweenadministrations of about one week to about four weeks.
 32. The methodaccording to any one of claims 1-31, wherein the modified T cells fromstep (a) are administered concurrently or sequentially with the modifiedcells from step (b).
 33. The method according to claim 32, wherein theinitial dose of modified cells from step (b) are administered from about1 day to about 28 days after administering the modified T cells fromstep (a).
 34. The method according to claim 32, wherein the modifiedcells from step (b) are administered sequentially within about 24 hoursof administering the modified T cells from step (a), or the modified Tcells from step (a) are administered sequentially within about 24 hoursof administering the modified cells from step (b).
 35. The methodaccording to claim 32, wherein the initial administration comprisesadministering a composition comprising a mixture of the modified T cellsfrom step (a) and the modified cells from step (b).
 36. The methodaccording to any one of claims 32-35, wherein the modified cells fromstep (b) are further administered in one or more doses at one or moreintervals for up to about 365 days after administering the modified Tcells from step (a).
 37. The method according to any one of claims32-36, wherein the modified cells from step (b) are modifiedhematopoietic progenitor cells
 38. The method according to any one ofclaims 32-36, wherein the modified cells from step (b) are modified Tcells.
 39. The method according to any one of claims 1-38, wherein themodified T cells from step (a) are administered to the subject at a doseof about 10⁶ cells/m² to about 10¹¹ cells/m² and the modified T cellsfrom step (b) are administered to the subject at a dose of about 10⁶cells/m² to about 10¹¹ cells/m².
 40. The method according to any one ofclaims 1-39, wherein the method further comprises administering acytokine.
 41. The method according to claim 40, wherein the cytokine isIL-2, IL-15, IL-21 or any combination thereof.
 42. The method accordingto claim 41, wherein the cytokine is IL-2 and is administeredconcurrently or sequentially with the modified T cells from step (a).43. The method according to claim 42, wherein the cytokine isadministered sequentially, provided that the subject was administeredthe modified T cells from step (a) at least three or four times beforecytokine administration.
 44. The method according to any one of claims40-43, wherein the cytokine is IL-2 and IL-2 is administeredsubcutaneously.
 45. The method according to any one of claims 1-44,wherein the subject is further receiving immunosuppressive therapy. 46.The method according to claim 45, wherein the immunosuppressive therapyis selected from calcineurin inhibitors, corticosteroids, microtubuleinhibitors, low dose of a mycophenolic acid prodrug, or any combinationthereof.
 47. The method according to any one of claims 1-46, wherein thesubject has received a non-myeloablative or a myeloablativehematopoietic cell transplant.
 48. The method according to claim 47,wherein the hematopoietic cell transplant is autologous.
 49. The methodaccording to claim 47, wherein the hematopoietic cell transplant isallogeneic.
 50. The method according to any one of claims 47-49, whereinthe subject is administered the modified T cells from step (a) and/orthe modified cells from step (b) at least three months after thenon-myeloablative hematopoietic cell transplant or at least two monthsafter the myeloablative hematopoietic cell transplant.
 51. An adoptivecellular immunotherapy composition, comprising a population of modifiedhuman hematopoietic progenitor cells, modified human immune system cellsor a combination thereof, wherein a first population of modified cellsare T cells comprising a nucleic acid molecule that encodes an antigenbinding protein, and a second population of modified cells comprising anucleic acid molecule that encodes the antigen, wherein the antigenbinding protein comprises a hydrophobic portion disposed between anextracellular binding component and an intracellular effector component,and wherein the extracellular binding component is specific for theantigen encoded by the second population of modified cells.
 52. Thecomposition according to claim 51, wherein the binding component is anantibody variable fragment (Fv), a TCR variable domain, a receptorectodomain, or a ligand.
 53. The composition according to claim 52,wherein the binding component is a scFv or scTCR comprising a variableregion linker.
 54. The composition according to claim 53, wherein thevariable region linker comprises a (Gly_(x)Ser_(y))_(n), wherein x and yare independently an integer from 1 to 5, and n is an integer from 1 to10.
 55. The composition according to any one of claims 51-54, whereinthe hydrophobic portion is a transmembrane domain.
 56. The compositionaccording to claim 55, wherein the transmembrane domain is a CD4, CD8,CD28 or CD27 transmembrane domain.
 57. The composition according to anyone of claims 51-56, wherein the intracellular effector componentcomprises an intracellular region of CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28,CD79A, CD79B, CD134, CD137, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM,ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2,Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or anycombination thereof.
 58. The composition according to any one of claims51-57, wherein the intracellular effector component comprises CD3ζ andone or more of CD27, CD28, CD134, and CD137.
 59. The compositionaccording to any one of claims 51-57, wherein the intracellular effectorcomponent comprises LRP, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, or Ryk.60. The composition according to any one of claims 51-59, wherein thebinding component is specific for α-fetoprotein (AFP), B7H4, BTLA, CD3,CD19, CD20, CD25, CD22, CD28, CD30, CD40, CD44v6, CD52, CD56, CD79b,CD80, CD81, CD86, CD134 (0X40), CD137 (4-1BB), CD151, CD276, CA125, CEA,CEACAM6, c-Met, CT-7, CTLA-4, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4,EphA2, FLT1, FLT4, Frizzled, O-acetyl-GD2, GD2, GHRHR, GHR, GITR, gp130,HVEM, IGF1R, IL6R, KDR, L1CAM, Lewis A, Lewis Y, LTβR, LIFRβ, LRP5,MAGE, mesothelin, MUC1, NY-ESO-1, a cancer-specific neoantigen, OSMRβ,PD1, PD-L1, PD-L2, PSMA, PTCH1, RANK, Robol, ROR1, TERT, TGFBR2, TGFBR1,TLR7, TLR9, TNFRSF4, TNFR1, TNFR2, tyrosinase, TWEAK-R, or WT-1.
 61. Thecomposition according to any one of claims 51-60, wherein the antigenbinding protein is a T cell receptor (TCR) or a chimeric antigenreceptor.
 62. The composition according to any one of claims 51-61,wherein the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4−CD8− double negative T cell, a γδ T cell, a natural killer cell, adendritic cell, or any combination thereof.
 63. The compositionaccording to any one of claims 51-62, wherein the T cell is a naïve Tcell, a central memory T cell, an effector memory T cell, or anycombination thereof.
 64. The composition according to any one of claims51-63, wherein the first population of modified T cells consistsessentially of CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ Tcells.
 65. The composition according to any one of claims 51-64, whereinthe second population of modified cells comprises modified humanhematopoietic progenitor cells.
 66. The composition according to any oneof claims 51-64, wherein the second population of modified cellscomprises modified human immune system cells.
 67. The compositionaccording to claim 66, wherein the modified human immune system cellsconsists essentially of CD4+ T cells, a CD8+ T cells, or both CD4+ andCD8+ T cells.
 68. The composition according to any one of claims 51-67,wherein the cells are recombinantly modified ex vivo.
 69. Thecomposition according to claim 68, wherein the ex vivo modified cellsare modified using a viral vector.
 70. The composition according toclaim 69, wherein the viral vector is a lentiviral vector or aγ-retroviral vector.
 71. The composition according to any one of claims51-70, wherein the population of modified cells is allogeneic,syngeneic, or autologous.
 72. The adoptive cellular immunotherapycomposition according to any one of claims 51-71, further comprising apharmaceutically acceptable carrier, diluent, or excipient.
 73. A unitdose form comprising a composition according to any one of claims 51-72.74. A unit dose form comprising modified T cells from step (a) and aunit dose form comprising modified cells from step (b) according to anyone of claims 1-50.
 75. The unit dose form according to claim 66,wherein the modified cells from step (b) are modified humanhematopoietic progenitor cells or modified human T cells.